ABSTRACT Abstract: Mass spectrometry imaging (MSI) provides unprecedented detail of molecular distributions across ex vivo tissue samples. Although MSI has the capability to map molecules ranging from large proteins to small metabolites, these data only describe the tissue status at a single time point and lack any information on how these molecules interact to provide metabolic function. Typical functional studies derive kinetic data from administration of isotope-labeled substrates and monitoring label transit in tissue samples harvested over a specified time-course. Isotope kinetics tracked by MSI, however, have some unique requirements to in order to perform a similar functional study and exploit the full power of the technique to provide location-specific metabolite flux. This proposal will develop an MSI-based method to track isotope metabolic activity through the use of a timed infusion of isotopologues of glycine to measure the rate of glutathione and serine metabolic flux within each 50 x 50 x 10 ?m image voxel. This timed infusion allows a voxel-by-voxel determinations of metabolic rates, i.e. functional images that are not possible by other methods. In addition, since metabolic flux and pathway selection is influenced both by tissue perfusion and oxygenation, we also will develop MSI-based methods to measure tissue perfusion and hypoxia in the same samples. Isotopologues of pimonidazole will be used to map regions of both chronic and cycling hypoxia that can be tied directly to glutathione metabolic activity. To demonstrate the feasibility of these techniques in different tissue types, we will map functional heterogeneity across mouse liver and mammary 4T1 tumors. As the MSI method uses frozen thin-sections, histochemical data from adjacent sections can be used to tie traditional tissue markers to metabolic function. The methods described herein will be demonstrated on the glutathione and serine pathways but can be used to study functional heterogeneity in any tissue and any metabolic network with proper selection of substrates.